SCIENTIFIC ABSTRACT There is an urgent need for more broadly effective cancer immunotherapies. The immune system can detect and eliminate cancer cells. However, tumors arise and persist largely due to acquired adaptations that enable tumor cells to avoid immune destruction by restricting innate immune surveillance, limiting cytotoxic T cell activity, and promoting overall immune tolerance. CD47 is a ubiquitously expressed signaling receptor that binds to its counter-receptor SIRPa on macrophages to inhibit phagocytosis. While this interaction is fundamental for physiological immune tolerance of healthy cells, it is used by tumors to evade innate immune surveillance. A growing number of hematological and solid tumors are known to express elevated CD47 levels, which is correlated with a worse prognosis. Elevated CD47 expression is also associated with cancer stem cell/tumor initiating cell populations that are refractory to treatment and a source of tumor recurrence. Preclinical studies using CD47 blocking antibodies, SIRPa decoy receptors, and antisense in xenograft and syngeneic mouse models show that disrupting CD47-SIRPa interaction leads to primary tumor elimination and inhibition of metastasis via activation of BOTH innate AND adaptive anti-tumor immune cells. CD47 is a potentially transformative target for cancer immunotherapy in a wide range of tumors. Currently, monoclonal antibodies and a SIRPa-Fc decoy receptor targeting CD47 are now in early stages of clinical development based on the compelling body of preclinical results and human expression data. We propose to develop small molecule inhibitors targeting the protein-protein interaction between CD47-SIRPa as new chemical probes to further elucidate the role of this axis in cancer biology and to identify new potential anti-cancer immunotherapeutics. In collaboration with NCATS and NCI we implemented a primary biochemical high throughput screen for CD47-SIRPa along with orthogonal confirmatory and counter screen assays that were validated in pilot screening of ~100,000 compounds from the NCATS chemical collection. Together with the use of biophysical and structural analysis we identified 7 high quality hits. We also established cell-based competition binding and phagocytosis assays to measure functional inhibition in order to prioritize the hits for optimization using medicinal chemistry. The goals of this proposal are to 1) screen the remaining NCATS chemical collections to substantially expand the chemical space explored and identify novel active chemotypes; 2) rank-order confirmed active compounds via biophysical characterization; 3) test biological efficacy and mechanism of action using cell-based assays for hit selection prior to medicinal chemistry optimization of selective CD47-SIRPa probes and therapeutic lead candidates. Successful execution of this project will yield validated probes to study the contribution of the CD47-SIRPa immunomodulatory pathway in tumor development and metastasis, and lead candidate compounds for the creation of novel cancer therapeutics.